Insulin stimulates a variety of primary and secondary events in target cells. What the signals are and where they are generated remains unknown. This proposal addresses these questions by investigating insulin action with a unique inhibitor of insulin- stimulated glucose transport, phenylarsine oxide, a trivalent arsenical which binds neighboring dithiols. The site of action of this arsenical appears to be post-receptor and provides a tool for identifying components of an insulin cascade. Using the exquisitely insulin-sensitive 3T3-L1 adipocytes, the primary goals of this research are as follows. 1) To determine the effect of PAO on insulin-activated glucose transport. Two theories of transport stimulation exist: one suggests that the transporter is "activated", the other suggests that the transporter is "recruited" from an intracellular site. By analyzing the effect of PAO on transporter distribution, discrimination between these two processes may be possible. 2) To identify common elements of insulin action. Several pathways demonstrate insulin sensitivity with differences in sensitivity and time-dependency. By comparing the effect of PAO on these different pathways, the groundwork will be laid for identifying common elements of insulin action. 3) To determine if the inhibitory effects of PAO are a result of intracellular or extracellular interactions. By using a PAO- affinity resin to affect transport in situ, we will determine if PAO entry into cells is a necessary component of its inhibitory effect. 4) To identify PAO-binding proteins. Using affinity chromatography, we propose to identify PAO-binding proteins which exhibit insulin-sensitive phosphorylation or activity. 5) To determine the time and insulin-concentration-dependence of inhibition of PAO-binding proteins. The temporal connection between activation of the insulin-receptor kinase and the glucose transporter suggests that an intermediate of insulin action must as a minimal criterion occur between these two events. With identification of potential intermediates this will be accessed along with sensitivity of the PAO binding proteins to insulin.